Infection with Chlamydia trachomatis is responsible for significant morbidity throughout the world. Chronic genital infections with C. trachomatis can lead to pelvic inflammatory disease, infertility, and other complications in women. Our overall goal in this grant is to define how C. trachomatis interacts with and manipulates the mammalian host during infection. In the previous project period we focused on a large-scale forward genetic screen in mice to map variant alleles that affect resistance to C. trachomatis. We identified a family of Immunity-Related-GTPases (IRGs) as responsible for mouse resistance to C. trachomatis. However in humans, IRGs are not responsible for C. trachomatis resistance; instead humans resist infection through the expression of indole-2,3-dioxygenase (IDO). The identification of IRGs and IDO as the key differences between mice and humans with regard to IFNg-mediated resistance leads us now to propose the development of a mouse model that mimics human infection with C. trachomatis. To achieve this goal we will engineer strains of humanized mice in which the mouse-specific immune response driven IRGs is replaced with the human response that depends on IDO expression. Currently, no small animal model exists that recapitulates the chronic disease as it manifests itself in humans, particularly the prolonged or recurring infections that cause pathologies of the genital tract and infertility in humans. We have also shown that a number of C. trachomatis protein effectors are translocated into the host cell cytosol during infection where they cleave or otherwise alter host proteins. Because alteration of host cell protein stability appears to be a general strategy used by C. trachomatis, we are applying two novel screening methods called stable isotope labeling with amino acids in cells culture (SILAC) and Global Protein Stability (GPS) to analyze, on a global scale, which host proteins are perturbed during infection with C. trachomatis. Once we have identified host proteins stabilized or destabilized during C. trachomatis infection, we will test whether reducing or increasing the prevalence of these proteins in cells impairs C. trachomatis development. Although pathogen-induced alterations of the host are a key to pathogenesis, it has not previously been possible to simultaneously assess the impact of infection on individual host proteins at the scale now possible with these approaches. The methods explored in this application allow, for the first time, an appreciation of how bacterial infection globally regulates host cell proteins and pathways beyond the transcriptional level. Understanding the interaction of C. trachomatis with its human host requires a large-scale approach to catalog the changes C. trachomatis induces in host proteins during infection. Once these proteins and the pathways in which they act are understood, the impact of disrupting these Chlamydia-induced manipulations can only be appreciated using small animal models that accurately reflect the pathogenesis of human C. trachomatis infection.